2G12 is a rare broadly-neutralizing antibody which binds to the HIV envelope glycoprotein gp120 by recognizing a cluster of carbohydrates. The goal of this project is to design a good structural mimic of the 2G12 epitope and test its ability to elicit a focused 2G12-like antibody response that can neutralize and protect against HIV infection. In Specific Aim 1, we will approach this problem from the standpoint of rational design. We have designed a tunable peptide scaffold for presentation of carbohydrates which can be used to present 2-6 carbohydrates with systematically varied inter-carbohydrate distances. We will synthesize a range of possible di-, tri- and tetravalent carbohydrate clusters this way, and compare their binding affinities for 2G12 by ELISA and surface plasmon resonance. These measurements will guide the refinement of our design. We will also study the effect of certain rigidifying cross-linkers on the synthetic antigen's ability to bind 2G12. In Specific Aim 2, we will approach the problem from the standpoint of directed evolution. We will use click chemistry to attach carbohydrate-azides to a random mRNA display library of ~1013 different unnatural alkynyl peptides. We will then select from this glycopeptide library the best binders to 2G12. Because every peptide in the library is attached to its encoding RNA, we can amplify and/or diversify the selection winners by PCR. Translation and click-chemistry glycosylation will yield a second-generation library with an increased population of 2G12 binders. Increasingly stringent cycles of screening with 2G12 and amplification/diversification will give rise to high affinity glycopeptide binders of 2G12, which should be good mimics of the 2G12 epitope. The best mimics will be sequenced and synthesized on preparative scale for further studies. Biophysical measurements will asses the ability of the best 2G12 epitope mimics from Aims 1 and 2 to compete with gp120 for binding to 2G12. In Specific Aim 3, we will test the immunogenicity of the best Aim1/Aim2 antigens in a two-phase rabbit study. A small pilot study will test 5 antigens in small groups. A follow-up study will reexamine the best 2 antigens with larger groups, varying doses, and varying adjuvants. In both studies, animal sera will be screened for neutralization activity against a range of HIV viral strains. Additionally, sera will be tested for the ability to bind gp120, as well as several glycopeptide test antigens. Whether or not a neutralizing antibody response is raised, this research will shed light on the 2G12-gp120 interaction. Moreover, the glycopeptide evolution technique developed in Aim 2 will have numerous other applications in fields such as cell signaling, cancer biology, and virology, and so will have wide- ranging impact across disciplines. Although the effort to create a neutralizing antibody vaccine against HIV-1 has so far met with little success, studies of the HIV-infected population have brought into the spotlight several examples of broadly neutralizing antibodies. The focus of this project is to apply knowledge gained from the study of one such antibody, 2G12, to design a vaccine which elicits a neutralizing antibody response by mimicking the 2G12 epitope.